CA1175356A - Chlorhexidine salts and compositions of same - Google Patents

Abstract

Abstract:
The dinalidixate and diphosphanilate salts of chlorhexidine possess antibacterial activity. They exhibit synergism as compared with comparable concentrations of chlorhexidine and the respective free acid. Synergistic compositions comprising a mixture of a chlorhexidine salt with phosphanilic acid or a salt of phosphanilic acid, or with nalidixic acid, or a salt of nalidixic acid are disclosed. Dermatological compositions of such novel salts, and all such mixtures, are provided.

Description

TECHNICAL FIEhD

The present in~vention relates to antibacterial com-pounds of ~he ~ormula:.

~ NH NH
C ~ NHCNHCNH ~

(CH2)6 2X

/
Cl ~ NHCNHCNH

wherein X is selected from the group consisting of nalidixic acid and phosphanilic acid, and compositions containin~
same. The invention also encompases mixtures of chlor-hexidine salts and nalidixic acid or a salt thereof or phosphanilic acid or a salt thereof. More particularly, the invention relates to certain novel chlorhexidine salts, antibacterial dermatological compositions containing such saltsj and certain mixtures of chlorhexidine and nalidixic acid, nalidixic salts, phosphanilic acid or phosphanilic salts.

3L 17535i~ii The novel salts comprise chlorhexidine dinalidi~ate and chlorhexidine diphosphanilate. Preferably, the chlorh~xidine diphosphanilate is employed as a hydrate or partial hydrate.
BACKGROUND ART

Chlorhexidine, nalidixic acid and phosphanilic acid are known in the art. Moreover, as is shown in U.S.
Patents 3,960,745, issued June 1, 1976, and 3,855,140, issued December 17, 1974, certain chlorhexidine salts are likewise known. Such salts include the hydrochloride, gluconate, isethionate, formate, acetate, glutamate, succinamate, monodiglycolate, dime_hanesulphonate, lactate, di-isobutyrate and the glucoheptonate.

Polyhydroxycarboxylic acid salts of biguanides, such as, for example, chlorhexidine di-D-gluconate, are disclo~ed in U.S. Patent 2,990,425, issued June 27, 1961, as being highly soluble in water.

The oral antibacterial use of water soluble salts of chlorhexiaine, such as the gluconate, acetate, fluoride, d~hydrogen fluoride and the dihydrogen chloride, is disclosed in U.S~ Patent 3,976,765, issued August 24, 1976.
~ .
An oral antibacterial composition comprising a combination of dodecyl-di-(aminoethyl)glycine and chlorhexidine or its digluconate, diacetate, dichlorid~ ¦
or monofluorophosphate salts is disclosed in U.S.
Pa~en~ 3,932,607, issuea January 13, 1976.

1~7535~

- Salts of chlorhexidine with certain sequestering amino carboxylic acids are ~isclosed in U.S. Patent 3,888,947, issued June 10, 1975. Preferred salts include, mono-chlorhexidine nitrilotriacetate, tri-chlorhexidine di-[diethylene triaminepentaacetate], mono-chlorhexidine-di-~N,N-dihydroxyethylamino-acetate], mono-chlorhexidine N-hydroxyethylenedi-aminetriacetate and mono-chlorhexidine di-[~-hydro~yethylethylenediaminetri~cetate]. Such sequestrates are disclosed to have greater antibac-terial activity than the corresponding bisguanido salt (con~er column Y, lines 22 through 42 of the patent).

Bis-guanide hydro~yalkane sulphonic acid salts are dlsclosed in British Patent Specification 815,800. Such salts (including the isethionic acid salt, the 2:3-dihydroxypropane-1-sulphonic acid salt, tha 3-chloro-2-hydroxypropane-1-sulphonic acid salt and the 2-hydroxypropane-1-sulphonic acid salt) are asserted to advantageously possess high solubili~y in water.

U.S. Patent 3,152,181, issued October 6, 1964, discloses monobiguanides having at least one alkoxy-propyl group having from aboul 11 to l9 caxbon atoms attached to the Nl or N5 terminal nitrogan atoms.
Such compounds are said to display exceptional antimicrobial activity and may be employed as the free base or, where water solubility is a factor in their use, as their salts with the inorganic and organic acids (such as mono and polycarboxylic and sulfur-containing mono and pcly acids and acidic nitrogen compounds). E~emplary of acid salts are .. . . .
, . . .

~L175356 the hydrochloride, hydrobromide, sulfate, phosphate, borate, phosphite, sulfite, sulfonate, nitrite, car-bonate, nitrate, acetate, tartrate, propionate, oxalate, maleate, malate, picrate and ~-ethoxypropionate salts.
Examples of suitable acidic nitrogen compounds are theophylline, substituted theophyllines and similar purines, saccharin, phthalimide, benzoxazine-2, 4-diones, oxazolidine-2,4-dione and substituted oxazoli-done-2,4-diones, N-p-methylbenzene sulfonyl-N'-n-butylurea, barbituric acids, mercaptobenzothiazole, 8-chlorotheo-phylline and succinimide. Patentees teach (at column 11, lines 36 through 70) that their monobiguanides can be employed ~Jith other medicaments.

As stated heretofore, nalidixic acid, phosphanilic acid and chlorhexidine are known in the art. The antibacterial agent, nalidixic acid (l-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8 naphthyridine-3-carboxylic acid) is the subject of U.S. Patent 3,590,036, issued June 29, 1971. Nalidixic acid has not to the instant inventors' knowledge been heretofore utilized topically.

Phosphanilic acid (p-NH2C6H4PO3H2, 4-aminobenzene phosphonic acid) has been synthesized (inter alia Doak et al. JACS 74 (1952)) and found to be active against 25 various organisms (see for example Kuhn et al., Ber. 75,711 (1942); Klotz et al., JACS 69,473 (1947~; and Thayer et al., ~ntibiotics and Chemotherapy, 3,?56 (1953)).

U.S. Patent 3,159,537, issued December 1, 1964, teaches that certain phosphonic acid compounds, including phosphanilic acid, increase the oral absorption (viz.
increase blood level) of tetracycline antibiotics.

.

117~i35 E;

, .. I
Complexes of phosphanillc acid and an amino- I .
acridine compound ~prefera~ly, . 9-~mino, 3-amino or 6-amino acridine) are disclosed in U.S. Patents 3,694,~47, issued S~pt~mber 26, 1972 and i,794,72i, issu~d F~bruary 26, 1974, as hAvin~ antibact~rial and antifungal activity.

Phosphanilic acid has also ~een reported to show synergistic action with trimethoprim against a variety of bacteria (see U.S. Patent 4,125,610, issued November 14, 1978). It has also been reported to show synergistic action wi~h neomycin and with streptomycin against Enterobacteriaceae (see Ciencia (Mexico) 17, 71-73 ~1957)).

Finally, it should be noted that the topical anti-inrective, chlorhexidine, (1,6-bis(N-p-chloro-phenyldiguanido)hexane) is long known to the prior art having been disclosed in U.S. Patent 2,684,924, issued July 27, 1954.

DETAILED DESCRIPTION OF INVENTION

The novel antibacterial compounds of the formula - ~ NH NH
Cl ~ \ / ~ NHCNHC~H ~ .
(CH2)6 . 2X
NH NH
Cl ~ NHCNNCNH /

wherein X is selected from the group consisting of nalidixi~ acid ~nd ph~phanilic ~cid are readily prepared by for example reacting ~75356 the desired acid, dissolved in suitable solvent therefor (preferahly hot ethanol in the case of nalidixic acid and hot water in the case of phosphanilic acid) with chlorhexidine free base, dissolved in a suitable solvent, preferably hot ethanol or hot m~thanol.

The resultant precipitate is recovered, washed then recrystallized according to methods known per se whereby the desired novel salt of the present invention is obtained.
In one aspect of the invention there is provided a composition for inhibiting growth of a bacterial species comprising a mixture of ~a) a chlorhexidine salt and (b) phosphanilic acid, a salt of phosphanilic acid, nalidixic acid, or a salt of nalidixic acid; components (a) and (b) being present in the mixture in respective weight percent-age ratios such that the mixture has a synergistic antibacterial effect on said species but with the exclusion of the ratio 35:65% of (a) to 65:35% of (b).
In a further aspect of the invention there is provided a method for inhibiting the growth on an inanimate substrate of microorganism sensitive thereto comprising the ;~ step of contacting the microorganism with an amount suffici-ent to inhiblt its growth of the above composition.
The following examples more fully illustrate the method of preparation of the novel salts of he present inventiGn.

:

~ .

`:` \
~:IL753~i6 A solution of 505 mg. (1.0 mmole) of chlorhexidine fr~e base in 50 ml. hot ethanol is cautiously added to a hot solution of 464 mg. (2.0 mmole) of nalidixic acid in 50 ml. hot ethanol. An immediate precipitate results and is filtered off af~er the mixture has cooled. The,recovered precipitate is successively washed with ethanol, chloroform then ether. The washed solid precipitate is then recrystalli7ed from,dimethylformamide (DMF) whereby 0.6 g. (represent-ing a yield of 61.9~ of theoretical) of off-white solid chlorhexidine dinalidixate having a melting point of 224 to 226C is obtained. IR (K~r disc):
bands at 3330 - 2860 (broad multiplet), 1625, 1492, 14~5, 1252, 1132, 1092, 820 and 745 cm 1, :
: :
:: ~: : :

:

~.".~ ' ~17~3~i~ii 1 Anal. Calc'd for C46H54C12N14O6 C, 56.g6;
7I, 5.61; N, 20.21; Cl, 7.31; O, 9.90 Found: C, 56.86; ~, 5.74; N, 20.21; Cl, 7.25.

A warm solution of 505 mg. (1 O mmole) of chlorhexidine free base in 50 ml. methanol i~ add~d to a solution oF 346 mg. (1.0 mmole) of phosphanilic acid in 250 ml. of hot water. The resultant solution is then evaporated to approximately 50 ml. and cooled whereby a waxy solid separates. The waxy -solid is filtered off, washed with water then re-crystallized ~rom water. After drying, 205 mg. of chlorhexidine diphosphanilate dihydratej having a melting point of 172 - 174C, are produced. This represents a yield of 23.1~ of theoretical.A EtOH= 255 (Am = 51,600) IR (KBr disc): bands at 3460 to 2930 (broad multiplet~, 1650 to 1600 (broad multiplet), ~; 1515, 1490, 1420, 1130 and 828 cm 1.
;~: ' . .
;~ ~ Anal. Calc d for C34~50C12N12 ~ 2 C, 45.99;
~, 5.67; Cl, 7.g8; N, 18.93; O, 14.42; P, 6.97.
Found C, 45.94; H, 5.75; Cl, 8.25; N, 19.00; -P, 6.85.
~ , "' ' ' ' ~; .

I
~' ~753~;
~3 .

Chlorhexidine dinalidixate and chlorhexidine diphosphanilate produced in Examples 1 and 2, were tested for their activity in vitro against 32 strains of Pseudomonas aeruginosa, 26 strains of other gram- !
. . _ . , I
negative organisms and 18 gram-positive organisms.
Minimum inhib~tory concentration data for the reference standards, chlorhexidine digluconate, chlorhexidine diacetate, nalidixic acid and phosphanilic acid, were also obtained. Chlorhexidine base could not be employed as a reference as it is unstable. The results of the study are reported in Table I as follows:
.,'''' ' ' - i .
., , ' ' ' , . I

: .

~75356 , ~-~
c ~ o ~ ~

U
X ~ tO ~ ~ ~ ~ `J
_ ~
Z AA ~A AAA

_ _ '6 _ ~J ~
X 1~ A A A A A A ~
'_ 1 ~:

." .~ O _ .0 --~O ~ ~ ~O ", ~D _ _ _ _ _ _ _, ~ C_ 6~ .~ A A A A
~ O l ~ X .'.~

I ~! o l Ei O ~ I~
~ : q .:r O ~ o ~o ~ 0 ~ ~ ~ 1~ ` ~ ~

.~ -'S
X I ~ ~ co .~
~ ~00~ 0~ .2 o or~ 6~
~: :, _ C
~ 6~ 6C

_ . 6~ C
13'~ + + + ~ + + I I ~ I I I I I I I ~80 ~ 8 ~ V V l ~ 0 ' ' ~ ~ ~ .~ ~

_ _ X

~ ~53~ii6 The data of Table I shows that the dinalidixic acid salt of chlorhe~idine is more effective than either nalidixic acid or chlorhexidine alone as against many of the strains tes~ed. The data shows chlorh~xidin~ dinalidixate to be particularly activ~ against all strains of P. a~ruginosa as w~ll as against individual strains of K. pn~umoniae, E. cloacae and P. morganii while chlorhexidinè di phosphanilate is seen to be effective against 26 out of 32 s~rains of P. aeruginosa and a strain of P. mirabilis and P. vul~aris.

The results of Table I indicate that the nalidixic acid salt of chlorhexidine is more efEective than either of its two components alone.

To more fully investigate the synergistic effect of the novel salts of the present invention, chlorhexidine dinalidixate and chlorhexidine diphosphanilate, as well as nalidixic acid, phosphanilic acid, chlorhexidine diacetate and a 1:1 mixture of chlorhexidine digluconate and nalidixic acid, were tested for activity in vitro against 30 strains of Pseudomonas aeruginosa, 23 strains of other gram~negative organisms. Minimum inhibitory concentrations of the novel salts of the instant invention as well as the reference standards (viz.
chlorhexidine digluconate, chlorhexidine diacetate, nalidixic acid, phosphanilic acid and the 1:1 mixture of chlorhexidine digluconate and nalidixic acid) were obtained. In contrast to the prscedure employed in ~:1753~

obtaining the results of Table I, all compounds we,re tested on a straight weight basis and in a medium low in antibiotic antagonists (viz. Mueller~
Hinton Broth + 1~ Ionagar), thus increasing antibiotic sensitivity.

The results, as reported in Table II'which follows, show chlorhexidine dinalidixate to be more effective than either chlorhexidine or nalidixic acid, particularly against strains of Klebsie~la pneumoniae, and Enterococcus cloacae and 28 of 30 -- - ............. _ I
strains of P. aeruginosa. The phosphanilic acid salt of chlorhexidine 'shows ciear improvement over phosphanilic acid and chlorhexidine alone as against !
4 of 30 strains of P. aeruginosa. The results also show chlorhexidine diphosphanilate to be a very effec~ive antipseudomonal. It inhibits 29 of the 30 strains of P. aeruginosa at MIC values of 8 ~g/ml or less as compared to chlorhexidine digluconate and diacetate, which inhibit only 2 strains below 8 ~g/ml.

~1~53~6 _ _ _ . ... _ ,u N ~ ~ In o r A A A A A A A A A

U_.___ . ___ X 3 ~ m U"~ m n U~ o O ~ O r~ ~ ~

a . _ .. __ . ... __ C ~ 3 ~ ~ ~ ~ ~ CO
U 'Q O ~ 1 0 ~ CO _ N C~
.r o_~, e ~ o ~ r ~ 3 O O ~i ~O ~ 1 0 e _ .~~ 3 ~ ~1 .~
P~ o '~ e o o ~ ~o ~ ~ o ~ ~ ~ o ~ ~ ~ ~

U -- C~._ _ . __ _ A _ ... ___ _ ______.____ ~ (I) C) 3 o ~ . Vl ~n 00 o ~ r ~ ~ ~ ~ ~ O ~
~ .... _ : ~C u~
~ +~ ~ ~.1 m ~ m Q Z 3 A r~ ~o ~ ~ I N _ _< ~ o 11~
_ ~ e ~ ~ ~ ~o ~ ~ _ ~ .
~9 a ~ e e 3e cu~ 3 e o .~ ' ~ u~ o ~ 9 ~ 0 ~ u~ u, U~ 0 1:1. CL P ` .f ~ ~ --U ~. V ~ :~
~ ~ . ____ U~ V, t4 U~ ~

~l753~6 _ _ U N N
. ~O N ~D ~O

,U _ ~ 3 ~o ~o ~o N ~0 N ~O

R Ul ~ ¦ V
c o ~ ~ .:r N ~O CO CO ~D C
.e _ ~I C~l~

O V ~ ~ ~ N ~O C7~ C ~O U
~ ,C Cl~ ~O 1~ X
P ~ r .::1 N CO ~O N _ N It~ ~J
1-~o r _ O O
~ e ~ ~ - O

~ N ~ CC~ CO _ ,'.
~ ; I
e u ,~ ~ o ~ o : 1:1 Z C CO CC~ CO _ _ _ _ ~ r~ ~
~ ~ : '3~ ,.~ cnU~N ~ ~

~ ~: _ ~ ~
t~U~ l ~ ~
.~ ~
0~ ~0 0 ~::

'~

~753~ii6 To more fully amplify the unexpected synergism of the novel salts of the present invention the MIC
valu~s, reported in Tables I and.II in ~g/ml t were converted to MIC valu~s of ~ mol~s/ml using the following mol~cular formulae and molecular weights:

Chlorhexidine dinalidixate C~6H54cl2Nl4O6 .
969.94 Chlorhexidine d.iphosphanilate dihydrate . c34H50C12N128 2 1~ . 887.71 Chlorhexidine digluconate C34H54cl2Ol4Nlo 897.80 Chlorhexidine diacetate . .C26H38Cl2Nl0O4 625.56 ; 15 Nalidixic acid 12H12N23 23~.23 ~; Phosphanilic acld C6H8NO3P
. . 173.11 Tables III and IV show the results of such convers.ion : 20~ and enable a compariso~ of potencies on a more meaningful molecule to molecule basis rather than gram to gram basis.

.~ l ~1~7~ii3~i6 r~
~- ' ~OD ~ ~OG , oO~ loO c~ ~ o~ A ~A ~ ~'i Cl~ ~r O u~ O
A ~ A A A ~ A A A
_ _ .
~ 3 ~ ~ ~ A O ~ ¢~ CD A O O
~ A A A A A A A
,U _ ~

a. ~ ~.:r_ __o__~o~o __ ~
~ ~A eo _ _ 1~ _ O ~A "~ o ~, o o ~ O O NO ~0 C : A A A A A A

00r~
~ _o v~ ~ o U ~ _ ~0 ~0 W ~~ ~ r~ 15 o~ ~ ~ r~ O ~ ~D ~D ~D
S 3 ~ w ~ ~~ ~A _ O ~ A W ~ _ W 1 ~ ,~ _ ~ __ O ~ ~ ~D N 0~ A ul ~ ~ _ ~ o~
_ _ . ~o o~ D CO ~ W ~0 ~ ~ 51 _ ~ D O ~ ~.i ~ . _ _ ~: , ~ 5 ~ ~ ~ 9-~ ++++ ++ 1 1 1 1 1 1 1 1 1 1 :: _ _ _ a ~ 5 ~ ~ 5 ~
o ~ X I ~ u ~ cJ E 9 ~ ~ 9 ~ u~ ~ vi D~ r w ~
:~; ._ ~7~
.~

" ~ ~r~

u rl ~ U~
e ~O ~ O OO , u~ r~ O ~ o ~ O ~
~! o ~ A
.
U
X ~ A ~ Cl~ ~ ~ CO N ~ O ~i U~l :~ 1~ ~ rl ,~ C~ ~ ~
.~ ~ ~ A A A
.

rl O O IC N ~ I'i 0 ~ ~ ~ ~ 0 rJ ~ O r~ A ~
C
.~ C" , ~, X ~o C ~7 N ~
1 a ~ o j ~ ~ O ~ ~ ~ ~ O ~

o,., a t~
8 r~-` ~ 1 ~ o -p~ o~~d x ~o o~ o~ Ao~

C _ X
~ . ~ .o c~ 0 u~--~ ).1 r~ ~ ~ ~ O O ~1 ~ ~ ~ ~ O ~ .-1 N c~
~ ~,, Z
1 a ~
.~ O ,u ~ ~. o~ ~A ~ A 1~
. ~ a u ~ ~ ~`I ~ ~ ~ ~ N ~ ~ ~ O O; I~--,~ ,,~ ," ,,, .,~ u~ ~ ~ U, ~Q z cu ; ~ , _ u a z ~
~
~ ~ + ~ + + + ~ l l l l l l l l l . 3 ~ L' c ~ -ol El - ~ 3 ~

o ~ `' C ~ P. U~ C4 C

.

~53~ii6 The results of Table III clearly indicate that the no~el chlorhexidine dinalidixate and di-phosphanilic salts of the instant invention are potent and exhibit synergistic effect particularly against P. aeruginosa with the nalidixate salt being the most widely synergistic. The synergistic effect of chlorhexidine dinalidixate is clearly seen by comparison of ~he results of Table III for K. pneumoniae, E. cloacae, P. mirabilis, P. morganii, P rettgeri,- P. vulgaris, S. marcescens, P. stuartii .
and P. aeruginosa. For example, chlorhexidine di-nalidixate is seen to have an MIC of 2.9 as against two strains of K. pneumoni~ae and 8. G against one strain of this organism. The corresponding values for nalidixic acid are 48.7 and 68.9; while those for chlorhexidine digluconate and chlorhexidine diacetate are respectively 70.1 and 35.6 and 51.1 and 51.1.
.~., , ..
The novel chlorhexidine dinalidixate of the present invention is seen to display a most impressive synergistic effect as against P. aeruginosa. Against 26 strains of this organism,chlorhexidine dinaladixate exh-ibited an MIC of 50.1; against five strains of such organism, it exhibited an MIC of 43.2; while against one strain of such organism, it exhibited an MIC of 32 . 9. In contrast thereto, nalidixic acid exhibited against these strains, MIC values, in each case, of more than 539 while chlorhexidine digluconate and chlorhexidine diacetate exhibited, as against the same strains, MIC values of respectively more than 139, more than 139 and 139 an- mo-e than 200, 200 and 00. ~ ¦

~' I

!

1175~56 The synergistic effect of chlorhexidine di-nalidixate is also clearly demonstrated from the results of Table IV. Synergisim is seen as .
against K. pneumoniae, E. cioacae, P. mirabilis, P. rettgeri, S. marcescens, P. stuartii and P. aeruginosa.

R~f~rring again to Table III, chlorhe~idine diphosphaniLate of the present invention demonstrates marked synergism against S. viridans, P. miraDilis, iO .P. vulgaris, one strain of P. stuartii and numerous -strains of. P. aeruginosa.

The result.s of Table IV demonstrate the asserted synergism of chlorhexidine diphosphanilate as against one strain of P. rettgeri and numerous strains of P. aeruginosa . .

~ , The present invention also contemplates dermatological compositions comprising a novel chlorhexidine salt.of the instant invention and a derma~ologically acceptable carri~r therefor.

Sui~a~le compositions include creams, lotions, suspensions, emulsions, ointments and pastes, and . surgical scrubs. - . , : Compositions in which a novel chlorhexidine :
salt of the present invention is produced in such compositions, in situ,~are also within the scope : of the invention.

' ' ' ' . ~i .
"

~L7~356 .
The following formulations axe offered to illustrate the compositions of the invention.
Although a particular formulation utilizes one of the novel compounds of the invention, it-should be readily appreciated that any of thenovel compounds or, for that matter, a mixture of such compounds, could he employed in lieu ;
ther~of.
'' ' .
Formulations l thxough ~, set forth in Table V
which follows, are prepared according to the following general procedure.

In a suitably sized premix container, the ~tearyl alcohol is dissolved in the Petrolatum with the aid of heat and gentle stirring. The temperature is then adjusted to about 62 to 68C.
' The propylene glycol and about 99~ of the purified water are combined in a suitably sized, preferably jacketed, main mix vessel and sti~red until a homogeneous solution is obtained. The resultant solution is heated to 62 to 68C then subjected to high speed mixing (preferably utilizing a suitable propeller mixture such as the Lightning~model ARL air mixer or similar type apparatus). The Carhomer is then slowly added thereto. High speed mixing is continued until the Carbomer is complètely dlspersed tapproximately one hour). The sodium lauryl sulfate, sorbic acid and the amphoteric-9 are then added, while mixing slowly, and the resultant mixture is mixed slowlv until it is homogeneous.

, ~L7~i356 . ~ .

In a suitably si~ed container, the remainder of the water (about 1%) is heated to about 40 to 45C then the dried sodium phosphate is added thereto under rapid stirring. The stirring is continued until a clear solution results. The clear sodium phosphate solution is then added to the mixture in the main mix vessel while mixing slowly. Mixing is continued until a smooth semi-gel is formed. While mixing slowly, heating is initiated. The temperature is adjusted to about 62 to 68C.

The petrolatum and stearyl a cohol, constituting the oil phase, are then slowly added to the main mix vessel containing the ingredients constituting the aqueous phase. This addition is made while ~ixing the aqueous phase at high speed. Mixing is continued for a period of about 5 to 10 minutes then cooling is initiated (preferably by circulation of cold water in the outer jacket of the main ~.ix vessel). During this cooling operation, the mixture is subjected to slow speed agitation (preferably using a side scraping sweep agitator of the Groen type or the like).
The mixture is cooled to a temperature of about 25 to 30C whereby a finished base is produced.

A small amount of the finished base (an amount sufficient to produce a workable consistency; about 5% in the case of Formulation l; about 10% in the case of Formulation 2 and about 15~ in the case of For-~.ulation 3) is added to a suitably sized container. The ~753~S

noyel chlorhexidine salt of the present inventionis added thereto ànd mixed therewith (with the aid of a spatula or suitable mixer) until it is uniformly dispersed in the finished lotion base.
The dispersion is passed through a roller mill (preferably of the Asra type or the like~ at an appropriate setting to produce a fine non-gritty particle size. ~his procedure is repeated if necessary whereby a mllled concentrate is obtained.

The milled concentrate is then added to the remaining finished base and mixed therewith at -slow speed (preferably using a sweep agitator) for one hour or until the uniform dispersion is formed.
.

.7 117S3~i6 .

.

. ~ .
_ I o ' o o o ~ o o o In O
~ O, ~ O In O va 1~ .~ N O
~1 If) I N ~I L~ O O O O O O
3 ~ 1 . o O , O C O ~D O O O U~ O
~1 O O 1 ~ 0 ~ ~O ~1 ~I N O
~Y) I N ~J Lt~ . O O O O O O
a) -. o O O, O ~ O O O ~ O
~ _~ O O U~ O ~ 1 ~ ~ O
C) . r-i. I ~ t~l 11~ 0 0 0 0 0 0 , . r-l N r~l O ~ I
Z
!
, .-.
. ' ' ' W , ' i. ''''' ' $
~a . .
.~.

, o, ~ o ~ . . ,, ~ . ~ ~ ~ o ~
~ o 2 ~ ~1 o cn o ~
O r~

~ 5~ ~ V -~
5-~ h ::~. O ~ O O
E~ O O Q ~
O : S .C ~ 0 ~0 0 h : ~ a ~, ~ , . i ::
: ~ ' i , I

' ' ' ' .
.

1~7~;3~

Formulations 4, 5 and 5, set forth in Table VI which follows, are prepared according to the following general procedure:

The Peg-8 and the lactic acid are added to a, preferably stainless steel, pr~mix container of suitable size and mixed slowly until a homogeneous solution is obtained.

The petroIatum, m~neral oil, ~anolin-oil, cetyl alcohol, ~ydrogenated polyisobutene, Peg-40 s;tearate, benzyl alcohol and sodium laurylsulfate are added to a main mix vessel which is preferably of stainless steel and steam jac~eted (e.g. Groen ~lodel TDC/2-20 or the like). The mixture is agitated slowly (preferably using a Lightning air mixer or a similar type mixture equipped with a propeller type blade) and heated.
Mixing and heating are continued until all solids are melted. The temperature is adjusted to about 65 to 70C. Mixing is continued for about 10 minutes then heating and agitation are discontinued.
The mi~ture is then cooled (preferably by intro-duction of cold water into the outer-jacket) and slow sp ed mixing is initiated (preferably with a side scraping sweep agitator). The mix~ure is ~ 25 permitted to cool to about 40 to 45C during ; which time continuous mixing is maintained.

The solutio~ of lactic acid and Peg-8 i5 then added to the ingredients contained in the main mix vessel, said ingredients constituting the oil phase. The addition is carried out . .

117~i3~;6 slowly and with constant mixing. Mixing and cooling are continued until a temperature of about 25 to 30C is attained whereby a finished base is produced .. ,, 1~ I
A small amount ~f the finished base tviZ. an amount sufficient to produce a workabl~ consistency;
approximately 5% in the case of Formulation 4, approximately 10% in the case of Formulation 5 and approximately 15% in the case of Formulation 6) is added to a suitably sized mixing container (for example a Hobart Model A-200D or the like) and the chlorhexidine diphosphanilate is added thereto. The combination of the chlorhexidine ~diphosphanilate and finished base is mixed slowly until a fairly uniform dispersion of the salt results. The dispersion is passed through a roller mill ~preferably of the Asra type or the like) at an appropriate setting to produce a fine non-gritty partiCLe size whereby a milled concentrate is obtained.
. .
The milled concentrate is added to the ; finished base and mixed there~ith at slow speed (preferab~y employing a side scraping sweep agita~or, for e~ample of the Groen type or the like) for about one hour or until a homogeneous dispersion s obtained.

, ` I

~S3~i6 o o oO o o o o o~ o oooooooU~oo .
~D u~ ~0 ~ ~ ~ ~ . ~ O O O O
. . - ,.
~, . .
'~1 ~
a),oooooo'OOOOo 3O o o o o O o Ul ~1 0 0 ...........
p u~~ o o~ o o o o ~ '.
.~:
O o o O o o o o o ~ o UO O o. O O O O o O' O o ~1' O o o o o O o U7 -1 o o ..... ~ .....
~r ,~ o oo ~ O O O O, .
. '.
. . :
O
; . '.
In ' .
~ . :

Q $ O

0~ , O ~~I S
,,~ ' O
~: , O ~ O o g' ~ U
~ ~ , ~ .~ ~ o ~ ~ u ~
: E~ ~ I O :~ h I ~ ~ '~
o s ~ ~ o o o 1~ ~1 \

11'7~3~;~

Formulations 7 and 8, set forth in Table VII
which follows ! are produced according to the following general procedure:

The glyceryl ~leate/~ropylene glycol, Peg-7-~ydrogenated castor oil, sorbitan oleate, o~leoyl~ydrogenated animal protein, Arlacel 481, light mineral -~1, ~ydrogenated polyisobutene, lanoiin a lcohol/mineral Oil, caprylic~capric triglycerides, propyl paraben and beeswax are added to a, pre~erably stainless steel and jacketed, premix container of suitable size. The mixture is subjected to heating and slow mixing ~preferably with a Lightning Model ARL air mixer with propeller type agitator or similar apparatus). The temperature is adjusted to about 75 to 8SC whereby an oil phase is produced.

The water, iactic acid, propylene glycol and dried sodium phosphate are added to a main mixing vessel which is preferably of stainless steel and jacketed (~or example the Groen Model TDC~2-20 or similar apparatus). This addition is made while heating and mixing at moderate speed (preferably èmplo~ing a Lightnin Model ARL air mixer or the ke). Mixing is continued until a clear solution is formed, then the sorbitol! methyl paraben and - the ~itanium ~oxide are added. Mixing and heating are continued until a homogeneous mixture - is produced. The temperature is adjusted to about 75 to 85C whereby a ~ater phase is produced.
- , - ' ' . .

.. . ... . .
~"" ' - .

53Si~

When the wa,ter phase and the oil phase are at a proper temperature, the oil phase is added slowly to the water phase while mixing constantly at moderate speed (preferably employing a Lightnin S Model ARL air mixer or similiar apparatus) whereby an emulsion is produced. The ~agnesium stearate is added to the emulsion and mixed therewith for about 20 minutes at a temperature of about 75 to 85C. The heating is then discontinued (preferably by introduction of cold water into the outer jacket of the main mixing vessel). Slow speed mixing is initiated, preferably using a side scraping sweep agitator (such as the Groen Model TDC/2-20 style agitator or similar apparatus).
Mixing and cooling are permitted to continue until the temperature reaches about 25 to 30C whereby a finished base is produced.

~ small amount of the finished base (viz. an amount sufficiant to produce a workable consistency;
about 10% in the case of Formulation 7 and about 15% in the case of Formulation 8) is added to a suitably sized container. The novel chlorhexldine salt of the present invention is then added thereto and mixed therewith using a spatula or other suitable mixer until a fairly uniform dispersion of the salt in the ointment base results. This dispersion , is passed one or more times through a roller mill of suitable capacity (preferably of the Asra type or the like) at an appropriate mill setting to produce a fine non-gritty particle size wherPby a milled concentxate is produced.

~ ~7~3~S

The ~lilled concentrate is added to the remaining finished base and mixed therewith (preferably using a Groen sweep agitator or similar equipment) at slow speed for ahout one hour or until a homogeneous dispersion is formed.

~ ~ .
::

: ~ ..

~1~7~3~fi o C~ ~ o o o o o o o o o o o o Ul o ~ o o ,~: o o co o o oo o o o o o o o o ~1 N ~1 ,1 o tJ~ CO
.,_1I U~ ~ O O O OXi ~D C ~ ~ ~ ~ ~ ~ O O O 'O O
~1 ~ ~1 ~1` ~1 ` O
. ' . . ~1 ~

. o o ~ o o o o o o o o o o o o U~ o ,~ o o o o C~ o o o o o o o. o o o o o ~ ~ ,1 ~ o 5:: . ~ ................. .
~` ~ I ~ o o o o CO ~ ~ ~ ~ ~ ~ ~ ~ o o o o o a~ o ~Z; . , "' H

~ . .
a~
-E~
, ~ ' . O
O
-~
O
- a) a) o . s~
Q ~ u h :~ ~ ~ i ,~ ~ o o O U ~ O ~ ~ h : ~ O ~ ~ o C ,a ~ ' ~ ~ ~ h ~ ~ X ~ O >~ ~ ~ I
8 o o Q. s ~ I o ~ ~ u 4 o ~ ~ ~ Q~
u c~ u m ~ ~ fS u~ ~ ~n o , 1~7~356 The novel chlorhexidine salts of the present invention represent an unusual approach to the synthesis of superior antimicrobials. The data of Tables I through IV clearly demonstrate that these chlorhexidine salts possess unexpected and medically useful antibacterial properties.

Chlorhexidine is a known antimicrobial agent.
Attempts have been made in the prior art to synthesize the "best" salts of chlorhexidine for various applications.
However, prior art attempts in this regard have utilized an entirely different approach than that employed herein.
In the prior art approach, chlorhexidine sa~ts are made with materials which do not of themselves possess any significant general antimicrobial activity as compared to chlorhexidine. As would be expected, some prior art chlorhexidine salts have better activity or more desirable physical properties than other of such salts.
However, any improved activity is merely a fortuitous result of such synthetic approach. The approach used in the present invention viz. reacting two antimicrobial compounds to produce a new antimicrobial compound which is formed solely from the two "parent" anti-microbials is quite distinct from the prior art approach of making a simple salt of an antimicrobial or mixing two antimicrobial agents to produce a mixture having advantageous properties. For most combinations of antimicrobials, the formation of a useful antimicrobial compound from two known antimicrobials is not a feasible or rational means of seeking improved antimicrobial properties.
.

1~753~

The data of Tables I - I~ show that the novel chlorhexidine salts disclosed herein are surprisingly and unexpectedly superior to like concentrations of their compo~ents or like concentrations of simple 5 mixtures of their components. This superiority is seen in thatO

(a) The test data of Tables I - IV show that the chlorhexidine salts of the present invention, when tested against the specified bacteria, are free from undesirable antagonistic properties of a simple mixture of the parent compounds;

(b~ The chlorhexidine nalidixate and phosphanilate salts unexpectedly exhibit a broader spectrum of antibacterial activity than other chlor-hexidine derivatives;
.
(c) Chlorhexidine nalidixate and phosphanilate exhibit substantially better activity against the majority of strains of Pseudomonas aeruginosa, an organism commonly encountered in burn infections. This is an extremely important and novel activity. There has been considerable interest in the skin degerming activity of chlorhexidine derivatives and their ability to prevent burn infections;
however, prior art chlorhexidine compounds uniformly exhibit inadequate activity against many Pseudomonas aeruginosa strains The surprising activity of chlorhexidine nalidixate and phosphanilate against the majority of strains of Pseudomonas aeruginosa tested is 1 17535~
! 32 extremely important to the medical profession and constitutes a potentially significant advance in the treatment and prevention of burn infections.

The following analysis is offered in substantiation of points (a~, (b) and (c) above. Chlorhexidine nalidixate was employed since the test data of Tables I - I~ include the results of testing a 1:1 mixture of chlorhexidine digluconate and nalidixic acid.
.
; 10 Various methods have been employed to support a claim of synergism. The most desirable involve calculation of a numerical value for synergy to facilitate comparisons.
One such prior art recognized method is that of Rull et al, Applied Microbiology 9,538-541, 1961. This method of calculation has been used in support of several tpossibly many) United States patents, for example, U.S. patent 3,989,585 of Swered et al.

The following formula is used:

+ Q =~ Synergism Index Value of Synergism Index Indicates 1 Antagonism ~1 Additivity ~: :
~ <1 Synergism :: ~

~L17S3~

where QA~ QB = quantities of compounds A or B, respectively, in the mixture at the minimum inhibitory concentration Qa Qb = quantities of compounds A or B, respectively, acting alone to produce an end point.

The data of Table II were employed in calculating a Synergism Index for chlorhexidine digluconate and for nalidixic acid, as against ~arious organisms. The method of Kull et al was utilized and the results are shown in the following Table ~III.

:

..

~1 1 7 5 3~6 'TABLE' VI'II
Synergism Index for Chlorhexidine Digluconate - and NalidiX.ic Aci'a'from Data of Tab'le 'II
Compound A = Chlorhexidine Digluconate Compound B = Nalidlxic Acid QA QB A ~ B = Synergism Index Organism Qa Qb Qa Qb S. aureus 2.40 .02 2.42 (Antagonistic) S. pneumoniae4.90<.40 >4.90 (Antagonistic) S. pyogenes2.48 c.06 >2.48 (Antagonistic) S. viridans1.97 <.25 ~1.97 (Antagonistic) Streptococcus5.71<.06 ,5.71 (Antagonistic3 (B-hemolytic) S. faecalis2.83 C.O5 >2.83 (Antagonistic) E. CO1; .79 .25 1.04 (Add;t;Ye) K. pneumoniae.18. 25 .43 (Synergistic) -- .25 . 25 .50 ( Synergistic) E. cloacae 13 . 25 . 38 (Synergistic) -- - 25 .35 .60 (Synergistic) P. mirabilis.75 .25 1.00 (Additive) P. rettgeri.03 1.00 1.03 (Additive) .16 1.00 1.16 (Additive~
P. vulgaris1.004.00 5.00 (Antagonistic) S. marcescens .20 .96 1.16 (Additive) P. stuartii25 1.00 1.25 ~Additive) 1.00 1. 25 (Additive) P aeruginosa 1.00 .06 1.06 (Additive~
1 00 .06 1. 06 (Additive) 13 . 06 .19 (Synergistic) .25 < 03 ,.25 (Synergistic) 23 07 . 30 (Synergistic) 33 <.05 >.33 (Synergistic) .25 ' .06 .31 (Synergistic) ~1753~i~

It can be seen from the results of Table VIII ~hat for some organisms, particularly gram-positive cocci, the actions of nalidixic acid and chlorhexidine digluconate are strongly antagonistic. In other words, it takes a higher concentration of the mixture of nalidixic acid and chlorhexidine digluconate to inhibit the test organism than would be expected from the amount of each pure compound required to in~ibit the same organism.

It is further e~ident from the data of Table VIII
that cases o~ additivity also exist and for some organisms (particularly Pseud monas aeruginosa) the actions of nalidixic acid and chlorhexidine digluconate are syner-gistic.

For comparative purposes, the Minimum Inhibitory Concentration (MIC) of chlorhexidine nalidixate is compared to the MIC of chlorhexidine digluconate and to the MIC of a 1:1 mixture o chlorhexidine digluconate and nalidixic acid in the following Table IX. Table I~
also sets forth the Synergism Index for the 1:1 mixture of chlorhexidine digluconate and nalidixic acid as calculated and shown in Table ~III. To facilitate such comparison~ the MIC of the 1:1 mixture of chlor-hexidine digluconate and nalidixic acid was corrected to exclude the weight portion that is "digluconate"
~about 22%)~ The results are set forth in the column of Table IX labeled "Corrected MIC 1/1 Mixture". This weight correction would not be necessary i it were possible to test pure chloxhexidine; howe~erl chlor-hexidine base is unstable and any MIC data generated with it would be unreliable.

~ 1753~ 1 ' .~ ~ I
~ X.X , ,~
! ~ .
oz ~t ~
~XO ........ . . .. . . ~

~ O r~ ~ ~ c~ N 0~ l ~ c~ o~ ~O X
. ~ ~ ~ r~ r~ ~ ~ ~_~
' . ~al . -~ +~ O~ 00 ~ r~ ~ ~ r O~ . o .~ 0 a~ _QJ r - ~- ~ ~1 ~ ~ ~ V~
X r~t ~ O .-- t-H Et tJ~ ~ c~ Cl 1 t ~ r~ ~U ' t U~
P~ U _ .--.~ C~'t Ir~ ~ ~ d ~L~ u~ 0~l a>
~ ~, ~ .~ . - ~ w ~ ~o , ~ r~ ~ o u~ ~ ~ ~t ~ co co co t~ ca ~ ~ c I O ~ ~ cn .--~ ~ ~ >.
~ .,. V~
~ ~ _ z . E
~ ~ ,_~_ . r~

~ , o X ,, t~ W
~: ~ ._.,.. ,_., .~ ~ V ~ r~ U
~ ~ X ~ ~ ~> ~ ~ ~ r~ .,.. _., .,. ,~
t ~ v ~ ~ c c- s= ~ > ~ ~ c ~ ~ ~ ~ ~ ._.
O O O O O o ~ - O ~_ ~_ r- ~ ~ C ~ G~
cr) 1:0 cn c~ G ~ C c ~ ~ ~ ~ G~ ~ ~ ~ ~ s j a~~ 0 ~ ~ 0 ~ ~-o ~ C ~ r _ _ ~
¢CC¢a ~:c ~e~ ¢~ ¢':C¢Cl C~ ¢¢~ n ' . ' C

E W v~ C ~_ o .
tt ~ C a~ c ~1 ._ ~ - ~ i c - . i_ _1 c ~ u _ E~ ~ ~I G C

ttt 0 ~ ~ 1~ 1~ ~1 t;_ ~ I ~It~- ~1 ~
V~ ~1~ ~;~ ~IYI ~ ~I Qlt~ ~ _..
.

~7~3~i6 The data of Table IX clearly show that in those cases where the chlorhexidine digluconate/nalidixic acid mixture was determined to have a Synergism ~ndex indicative of antagonistic properties, the antagonism was eliminated by the synthesis of the chlorhexidine nalidixate salt. This is evident from a comparison of the higher MIC's of the mixture of chlorhexidine digluconate and nalidixic acid with the lower MIC`s of the chlorhexidine nalidixate salts.

The data of Table IX further demonstrate that in those cases where the mixture of chlorhexidine digluconate and nalidixic acid have a Synergism Index indicative of a synergistic result, the synergistic properties are preserved by formation of the chlorhexidine nalidixate salt.

It is surprising and unexpected that formation of the chlorhexidine nalidixate salt eliminates the antagonism obtained with a mixture of chlorhexidine digluconate and nalidixic acid.

~he data of Table IX show that chlorhexidine nalidixate has novel activity against Pseudomonas aeruginosa. MIC comparisons demonstrate that it is significantly better than chlorhexidine digluconate,which is available commercially and heretofore generally recognized as the best available chlorhexidine derivative.

3~

In summary, the data o Tables VIII and IX show that chlorhexidine nalidixate surprisingly and unexpectedly eliminates the undesirable an-tagonistic properties of a comparable mixture of its components (viz~ a 1:1 mixture of chlorhexidine digluconate and nalidixic acid) while preserving desirable synergistic properties and providing a unique and useful broad spectrum o~ activity.

MIC tests were carried out on a 1:1 mixture of chlorhexidine digluconate and phosphanilic acid. These MIC tests were conducted in the usual manner on solidified Mueller-Hinton medium. The method employed corresponds to that employed in generating the data of Tables I - IV.

The following Table X sets forth the test results.

~L7~;3~i~

.~ o~
S~ -- a)N L~ Ltl ~n Ltl LO U~ Ln ~ D L~l U'7 00 ~57 r~ 1 r-- ~
u~ ~ A A A A A A ~ '~ ' Y
O -~ Q

Q~ ~
~ 3 x 5~ ~ .- ,~, u~ a~ ,_ 1~ t~) '1' '~ ~3 S ~ S_ ~ ... . . . . ..
S. O '{:) O ~-- ~D d C~l ~ 00 ~ d' N 0~ N (r~ Ot) CC) O ~:: ~ c_ r - Q t-o ~ Q E
. ~ --o a~-~ E
~ ~t:: ~ a s a ~r ~; . .
r~ .~~0 N CO CO ~ d~
xa) o ~ aJ . . . . . . ..
.C O ~:: _ ~ r--~ O N C~l CO C~J ~ C~l ~1 ~- ~ N 00 ~0 D ~D r--~ CO 00 IJJ ~ ~ ~ ~ ~ ~ ~ ~ r-- ~ r_ m.-- ~ o x ~C_~ ~_.,_ E
a) ~$
X C- ', ~ ~
a~ ~0 ~-- V N e;l- ~0 r a~ i O N CO 00 C~l rO r~
.

(-- --~ ~ ~ d r~ N ~1 ~ v-- r~ C~J r--r~ ~ r~ ~ O r~
.- ~ v~ a~ ~
*f~ o ~ ¦ E ~ ~ O ¦ E ~ U `~I

., _ ~IJ o o ~ ~ - ~) o _ ~ ~ - C ~
~ Q o -- E Q ~ o Q -- E o ~ E ~ a~
o ~ ~ ~ ~n ~ ~ ~ V~ 1 Cl~

~IL~t;~3~i~

The method of Kull et al, pre~iously referred to, was employed in calculating the Synergism Indices of the following ~able XI.

11753~

X
C ~, ~ ~ ,, ~ ~ .~ ., ., ., ., ., ., V ~ ~, E c~ ~n ~ ~ ~ ,- ~ Q~ , ~n O ~ O o O o o o cn o ~"
c7- t c_ ~ ~ ~ ~ c ~ C~ C ~ ~ t c C C 0 C~ ~ ~c ~ a~
C ~ ~ ~ ~ >, ~ ~ ~ ~ ~ C ~ ~ C C C C >, V~ _ ~
ll c~l o cn oo o ~ ~ ,~ ~ 00 Ln ~ ~ O n o . o ~ I
a:l ~:1 Lr) 1~ r~ O Lt~ ~ 0 1~ 0 1~ ~-- O N ~ ~0 O C~:) ~ 00 ~ ~D I
O' O' ... , . . .
~ A /~ /~ A A
cc ~ Oa 10~l ~ '`~ ~ ~ o o co oL.
X s V V V V V V V C~ C~ r~
a V) C s Q

U) ~C ~ ~ r~ 1~ o Lf~ O O 1~ 0 U~ g O g 8 ' g C~l ~ O O ~n o a) a~
C
X
~ ~ o a~ O ~ a~
C
o-, C
o _ C
X ~ ~
~ ~ ~ o :~: ~ C.~' o o ~ ~ ~ ~

'~ E~ ~' 71 ~ U~ U~

0 ~0 o o 1~7~3~;6 The data of Table XI show that mixtures of chlor-hexidine digluconate and phosphanilic acid exhibit ~arying properties against the different test organisms and that the pattern of synergism/antagonism differs from that of chlorhexidine digluconate/nalidixic acid mixtures. The chlorhexidine digluconate/phosphanilic acid mixture shows synergism or additivity against gram-positive cocci, and wide variation between antagonism and synergism against gram-negative bacilli. This is almost completely opposite to the synergism pattern shown by the mixture of chlorhexidine digluconate and nalidixic acid.
For most strains of Pseudomonas aeruginosa, the mixture of chlorhexidine digluconate and phosphanilic acid is synergistic or weakly antagonistic.

In the following Table XII, M¢C values for the compounds are compared to the Synergism Indices shown in Table XI. The novel and unexpected properties of the chlorhexidine phosphanilate salt are apparent from the data of Table XII. To facilitate comparison, the MIC of the mixture in Table XII has been corrected to eliminate the weight portion which is "digluconate", about 22%.
This was done because pure chlorhexidine base is unstable and cannot be tested for direct comparison.

' 753~i6 a~ a C~
.. .~
~) X C
O V~ r-- ~O ~t N ~- C~ D U ) N t~) 00 00 r~ O
S S

~ a~
., - C
X O
QJ ~ r--~_ I~ ~ r-- 00 ~ 1~ r~ ~
~: O C7 ~ ¦ o N ~.D d'C~l N C~ CO Ln CO ¦ C~l O C~l 00 CO t~l S ~I r~ v ~) ~ N . r- IS7'J a:~

X
a~
_~ co loo oo ;~
~_ ~ X 1~ 1~ ~ ) C~J o ~) N ,_ G O 00 . O ~) Lf~ O O 0~
~-~ . ... . . . . . .. . .. . . . . U) Q~ ~ ~ 1~ N Ln 0 N t~L~ Itl CO ~ 11'1 ~ 3 Lt'~ ) ~ U:l D ~
r ~;5~ ,_ ~ - N C~l~J C~J ~-- . c ~o r- o ~ ~

m X '~ n _ ~8 o o ~ Z - ~ ~ V . . o C! t CL cn~3 t3 i=
~ o e.~. ;:LO V ~c .~ r.~ ~ ~ r,~
a~ ~ ,~ ,_ .,_ r---~ C17 .~ N 00 00 ~) ~ ~ ~ ~ V
~ C) _O ~ s ~ o C~ N CO ~ J C~
i-- ~ ~ C~ C~l ~~ ~ I ~ ~) r- I V~
C O S' ~ ~ O .
U~ ~S _ ~ O S c ~)~r~ZS

~ ~ X o ~0 C ~ ~ ~ V V V V V V ~
~: E ~ ~ ,_ v v v ~ v ~ ._ . , , ~ v-, v) u~ X ~ ~ ~ ~ ~ ~ c~ V) ~ v~ v~
, ~-~ vl ~ u~ ~ a) v,r- a) ,--. ,_ ,_ , ~, v~-~ v~ I
s ., ,-,- ~ , > ~ -- ~ ~~ :~ c ~ ~ ~ ~ ~ ,- ~-,- l ~_ ~ cn , ~, ~ r--o o o o o o CJ) O C~
c _ ~ _ ~ ~ t ~ ~CO .~ ci~ r~ n r~5 r3 a~ n~ ~ v) r_ ~ C- ~ ~ C ~ ~ ~ ~ ~ ~ ~ ~ ~ -}~ ~ +~ ~ ~ ~i ~ ~) C ~ C
CC ~ C C ~ cl V~ ~
qc) E ~ ,," a~ -r .~ u~ u~ v v ~ ,- v~ ., v~ ~ ._ o r ~ c ._ ~ ., c a) ~ .~ ~ ._ v ._ ~ -a cr v~ O ~ rr,~ 1~ 0 ~-- O ~ .~ ~ ~ S. t~ ~ .
~ _ E ~ ~ ~ o ~ E v D rcS CJ ~5 QJ ~ G
o ~ a~ o s 'c~ ~ -I 3 ,cs ~ ~ c~ ~ as 3 :~ c ~ ._ E ~ ~ o l c ~ ., o Q~ ~s 3 ~ ~ . .
~S ~:L ~1 ::' a~ ~ 11 1~ I Q ~ E E ~. :~ E v7 ~ LLI

`" ~17~i3~1~

The data of Table XII clearly show that in cases where the mixture of chlorhexidine digluconate and phosphanilic acid had its strongest antagonistic properties (see the two cases underlined in Table XII), the antagonism was eliminated by synthesis of the chlorhexidine phosphanilate salt. This is seen from a comparison of the higher MIC's of the mixture with the lower MIC's of the chlorhexidine phosphanilate salt.

The data of Table XII further show that in cases where the mixture of chlorhexidine digluconate and phosphanilic acid was synergistic, the synergistic properties were preserved by the formation of the ~l chlorhexidine phosphanilate salt. What is more J
important for general antibacterial usage is the fact -that the MIC's for chlorhexidine phosphanilate are substantially better than the MIC's of the mixture of chlorhexidine digluconate and phosphanilic acid and the MIC's of chlorhexidine digluconate which is generally recognized as the best available chlorhexidine derivative.

It is important to note that the activity of chlor-hexidine phosphanilate against Pseudomonas aeruginosa is far superior to that of chlorhexidine digluconate in 30 of the 31 tested strains, and is equal for the remaining strain. It is evident that chlorhexidine pbosphanilate is an important antibacterial agent having potential use in the treatment of burns, where pseudomonal infections are commonplace.
' - ~L7~3~6 ~45-The data of Table II demonstrate the superior activity of chlorhexidine phosphanilate against Pseudo~
monas aeruginosa and gram-positive Staphylococcus and Streptococcus species. Phosphanilic acid gives MIC's >125~g/ml while chlorhexidine digluconate and chlorhexidine phosphanilate give MIC's from 0.25 -16~g/ml. From this data it may be concluded that, for these organisms, chlorhexidine phosphanilate is far superior to phosphanilic acid and as good as the best commercial material, chlorhexidine digluconate.

Referring now to the activity of chlorhexidine phosphanilate against gram-negative rods other than Pseudomonas: Phosphanilic acid MICIs are from 0.5 ->125~g/ml while chlorhexidine digluconates MIC1s are from 0.63 - 16~g/ml and chlorhexidine phosphanilates MIC's are from 0.63 - 6.4~g/ml. These data show that chlorhexidine phosphanilate is far superior to phosphanilic acid and better than chlorhexidine digluconate~

Referring further to the activity of chlorhexidine ~ phosphanilate against Pseudomonas aeruginosa illustrated ; in Table II: In screening antimicrobials for useful activity in media relatively free of antagonists (such as used in the tests employed to generate the results of Table II), 16~g/ml is the generally accepted cut-off point for useful activity. It is evident from the data that in all Pseudomonas cases, chlorhexidine phosphanilate meets this criterion while chlorhexidine digluconate is marginal or a failure against 93% of the tested strains. Phosphanilic acid fails by a significant ~l~S356 (fourfold) margin against 17% of the tested strains.
More importantly, chlorhexidine phosphanilate not only meets the cut-off criterion against 100~ of the tested strains, it surpasses the cut-off by a significant ~fourfold or better) margin as against 87% of the tested Pseudomonas aeruginosa strains.

The superior activity of chlorhexidine phosphanilate is also seen in Table III, particularly with reference to activity against gram-positive Staphylococcus and Streptococcus species. Phosphanilic acid gives MIC's >880 millimoles/ml while chlorhexidine digluconate and chlorhexidine phosphanilate give MIC's of from 0.3 to 17.8 or 18.6 millimoles/ml. From these data, one can conclude that, for these organisms, chlorhexidine phosphanilate is far superior to phosphanilic acid and as good as the best commercial material, chlorhexidine digluconate.

Turning now to the activity of chlorhexidine phosphanilate against gram-negative rods other than Pseudomonas (this includes aIl of the organisms of Table III except for Pseudomonas and the gram-positive cocci lviZ. Staphylococcus and Streptococcus] species).
Phosphanilic acid exhibits MIC's of from 3.5 to >880 millimoles/ml while chlorhexidine digluconate exhibits MIC's of from 0.7 to 17.8 millimoles/ml. In contrast thereto,chlorhexidine phosphanilate exhibits MIC's of from 0.7 to 7.4 millimoles/ml against the aforementioned gram-negative rods. It is clear from these data that chlorhexidine phosphanilate is far superior to phos-phanilic acid and slightly better than chlorhexidine digluconate.

;35i E;

_47_ Table III lends further support to the novel activity of chlorhexidine phosphanilate against Pseudomonas aeruginosa. As is seen in Table III, phosphanilic acid MICIs as against this organism range from 5.4 to ~880/millimoles/ml; chlorhexidine digluconate MICIs as against this organism range from 4.4 to 35.6 nillimoles/ml;while chlorhexidine phosphanilate MIC's as against this organism range ~rom 2.2 to 18.6 milli-moles/ml. These data make it abundantly clear that as against Pseudom~nas aeruginosa, chlorhexidine phosphanilate is significantly superior on a mole basis to phosphanilic acid and to chlorhexidine digluconate.

The results of Tables II and III clearly demonstrate the useful and novel properties of chlorhexidine phos-; 15 phanilate irrespective of whether the data is analyzed on a weight or molar basis.

:::

~: ::
' ~ ~

:

i3~i6 -~8-, Data analyzed previously in Tables VIII and XI indicate that 1-1 mixtures of chlorhexidine digluconate and nalidixic or phosphanilic acid have a synergistic effect against certain microorganisms. To elucidate the scope of this synergism, further experiments were conducted with additional compounds and at varying concentration ratios. The experiments tested chlorhexidine HCl, chlorhexidine digluconate, and chlorhexidine diacetate in combination with phosphanilic acid or nalidixic acid or the sodium or potassium salts of these acids. Mixtures were tested at weight X ratios of 5/95, 25/75, 50/50, 75/25 - and 95/5. The compounds in the mixtures were also tested individually. Tests were conducted on solidified Muel7er-Hinton medium in a manner similar to pre-vious tests except that the medium was solidified with Noble Agar rather than Ion Agar used preYious1y. The minimum inhibitory concentrations ~MIC's) ob-tained are reported in Tables XIII through XVIII. They are similar to MIC's reported in previous Tables but reflect some differences attributable to the use of different strains, slightly different test conditions, and more inten-sive replication.

For each mixtures MIC value in Tables XIII throu~h XVIII, a corres-ponding synergism index value can be calculated according to the method of Kull et al dexcribed previously. For example, in Tab1e XIII, the synergism index of a ~0/50 mixture of chlorhexidine digluconate and nalidixic acid agains~ 14 strains of Pseudomonas aeruginosa can be calculated according to the Kull et al formula as:
A ~ 52804) = 0.17 When all the individual synergism indices oorresponding to the mix-ture MIC's have been calculated, they can be averaged to produce mean synergism indices for each mixture ratio against all test organisms. This is a oonserva-$ive method of analysis because 1) each mean synergism index represents the overall results of at least duplicate tests against (using Table XIII as an example) 82 bacterial strains and 2) the method of Kull et al sets no theoretical upper limit on index value but restricts values indicating synergism to between ~L1753~i~

zero and 1Ø Therefore, one high index value indicating antagonism can outweigh several low index val~es indicating synergism, when an average is calculated. The mean synergism indices are displayed in Tables XIX and XX. Mixtures which display a consistent pattern of favorable synergism indices (<1.0) at different concentrations can be considered generally advantageous combinations for antibacterial applications. For example, the combinations of chlorhexidine digluconate with nalidixic acid or its sodium or potassium salts are synergistic at ratios from about 95/5 to about 5~95. Many combinations are synergistic only in narrower ranges. For example, chlor-hexidine digluconate potassium phosphanilate is synergistic at ratios from about 75/25 to about 25/75; chlorhexidine digluconate/phosphanilic acid from about 95/5 to about 25/75;
chlorhexidine hydrochloride/phosphanilic acid from about 95~5 to about 50/50; chlorhexidine diacetate/nalidixic acid from about 50/50 to about 5/95; and chlorhexidine hydrochloride/
potassium nalidixate from about 75/25 to about 5/95. Some mixtures lack general antibacterial synergism. Those tested which fit this category are chlorhexidine diacetate/~odium nalidixate, chlorhexidine diacetate/potassium nalidixate, chlorhexidine digluconate/sodium phosphanilate, chlorhexidine diacetate/phosphanilic acid, chlorhexidine diacetate/sodium phosphanilate, chlorhexidine diacetate/potassium phosphanilate and chlorhexidine hydrochloride/potassium phosphanilate.
Mixtures showing only limited general antibacterial value or ~ielding results difficult to interpret include chlorhexidine hydrochloride/nalidixic acid, chlorhexidine hydrochloride/
sodium nalidixate, and chlorhexidine hydrochloride/sodium phosphanilate.

::

1~75~56 -49a-It should be understood that combinations or mixture ratios which lack general antibacterial synergism may nonetheless have synergistic activity against individual species of microorganisms or under conditions different from those of our tests. For limited uses, such combinations or mixture ratios would be advantageous.

It should be noted that dermatological compo sitions of the synergistic mixtures disclosed herein may be readily prepared by adapting, if need be, the methods and the formulations disclosed herein for the novel dinalidixate and diphosphanilate salts of chlorhexidine of the invention.

~7S3~ j , TABLE XIII

Antibacterlal Actlvity o~ Chlorhex~dloe DiglucDnate 1D Comblnatlon uith Nalidlxlc J~cid (NA~ or lt8 Na+ aDd R+ Salt8 ~1 I C (IJg/~l )*
Drug Comblnations OrgaDl~m oi` Chlorhexldioe:Nalidixate (X:Z) Str~lDs 100:0 95:5 75:25 5~:50 25:75 5:95 0:100 Chlorhe~idlne NA P.aeruglnosa 14 27.6 11 11 8.4 14.1 6D 2250 DiglucDDate .Na+ 11 27.3 1513.7 6.2 16 61.I 2228 :}~ 13 2e 16 14.8 13.6 19.3 47,3 2243 ~NA ~. coli 2 1.4 1.2 0.84 1 2.4 3.4 22.6 Na+ 1 1.4 1.4 0.7 S0.5 1.4 4 16 .Kt 3 1.3 1.1 o.a 1.3 2.2 5 10.1 :NA R. PDeUmOniae 3 8 3.6 4 3.2 3.2 5.? 240.1 :Na~ 3 8 3.2 5 l.b 2.B 5 250.3 :RI 3 8 3.2 6.4 4 5 9 240.2 NA E. cloacae 211.3 8 2 ~.7 Z 5 7 16 :1~ 3 5.7 3.6 5 3.~ 4 6.4 232 Na-t p. stuar~ii 2 2 8 2 2.8 41.8 2 8 4 11.3 R~ 2 2.8 2.4 4 4.8 8 9.6 16 NA S. IDarcescens 2 132 6 7 6 7 5.7 8 Z-4 111 3 .g~ 2 16 S.7 ~1.3 ~.5 11,3 8 ~ 4.7 Na+ S. aureus 1 1 7 0-7 75 So 735 2 11.3 125 slt~ 2 0.84 0.7 0.5 0.5 1 ~i.7 ~177 A S. faecalie 2 1 4 1 1 510 42 2 4 16 2250

2 1.4 1.2 1.4 1.4 3.4 13.5 225D
i ;~ ~ *Geonetric ~ea~ value~ (of com'blnatlon8, ~here ~pplicable) f~om 2 exper~eDt8.

:':
I
?

`

- ~L17~i:3~

TABLE XIV

Antibacterlal Actl~lty Df Chlorhexldlne Diacetate ln Comblnatlo~
~lth Nalldlxic Acld (NA) Dr lt6 Na~ and ~+ Salt~
. - -M I C (~g¦~l)*
D~ug Co~b~natioDs Or~anls~ of Chlorhexidine:Nalldixate (Z:%) _ _ Stra~g 100:0 95:5 75:25 50:5D 25:75 5:95 0:100 Chlorhexldlne :~A P.aeru~inosa 2 3.4 4.8 3.4 1 8 ~2.6 22jo Dlacetate ~ 12 12 17 5 143 58 4 b 8 19 i25C
9 12.7 33.2 17.3 10.5 13.7 25.4 ~250 : :K~ 2 3.4 4.ô 4.8 5.7 6.7 19 ~2~0 12.1 21.1 14.4 13.5 13.9 19.7 ~250 :~A E. coli 3 0.5 0.4 0.6 0.5 1.3 4 10.1 Na~ 3 0.5 0.7 0.6 0.5 1.1 3.2 5.7 .g~ 3 0.5 0.5 0.6 C.6 1.1 2.~ 4 ~A R. pneumoniae 2 1.7 2.8 4 1.4 2.4 5.7 11.3 . 1 22.8 5.7 1.4 4 8 63 ~Nal 3 1.8 2.8 3.6 4 4 4.5 8

3 1.8 4 2.~ 3.6 4 4.5 16 NA E. cloacae 2 1.2 ~.7 4 0.7 2 4.8 11.3 : . 1 2 5.7 5.7 1 2.8 5.7 2177 ~Na~ 3 1.4 2.8 2.5 2.2 3.2 3.6 12.7 K+ 2 1.2 2.8 1.7 2.4 2.4 2.8 ~
1 2 8 4 4 4 444~9 :NA P. 6tuartll 2 2 2 2.8 2 5.7 11.3 22.6 : :~a~ 1 2.B 4 5.7 8 5.7 11.3 5.7 ~t 1 1.4 2 2 2 5.7 11.3 ~1.3 ~A S. r~rcescene 1 2 5.7 5.7 1.4 4 11.3 S.7 . 1 11.3 22.6 16 2.8 4 11.3 5.7 1 11.3 44 9 11 3 45 7 58~7 B 2 8 : ~ X~ 1 211.3 4 5.7 5.7 11~3 2 ' : 1 11.3 44.g 16 ~ 8 11.3 1.4 NA+ S. aur~us 3 0 5 0 8 0 6 0 6 1 6 3 2 ~ 177 a 3 0 5 0 6 0.7 0.7 1.1 2.8 288.9 A+ S. fsecalis 22 0~5 0~5 0 6 0 6 1 5 7 ~250 ~Na : 2 0 5 0 5 0.8 0.6 1.2 4 2250 _ ~Geomeerlc ~o valuc~ (of co~b~Da~lon~. ~here ~pplica~le~ fro~ 2 e~p~ri~ent~.

~ ' .
: ~ ~

, .

L7~3~i6 TABLE XV

Antlbac~er~al Actlvity of Chlorhexldlne Bydro2hloride in Coo~blnatio~
ui~h Nalldlx~Acid (I;A) or Its Na+ and ~ Salts = _ ~ . ~ I C (IJg/~l)~
Drug Co~binations OrganlslD of Chiorhex~d~De:2~alldl:~:ate (S:Z~
Stral~s100:0 g5:5 75:2S5û:5D25:75 5:g5 O:lOD
Chlc~rhexldiDe :NA P.AerugiDosa 5 67.5 42 95 88.7 2177 109 225O
~ydrochlorlde 9 2250 54.2 103.38B.7~177 103.3 ~250 :Ns+ 4 68.6 114.781.574.8 2250 250 ~250 9 2250 107.4 66.258.4225D 223 225o :~ 5fi7.5 51.4 63 88.75B.9 95 2233 ~ 8 225~ 60.4 74.888.75~.9 125 2229 :NA E. coll 3 2.8 2.5 6.4 3.6 10.1 5 20.2 :Na+ 3 2.8 3.2 2 6.4 3.2 2.8 16 :~ 2 3.4 85~7 ~.8 1.4 8 13.5 :NA l~.pneumonlae3 44.9 3.211.3 4.5 11.3 10.1 20.2 :Na~ 3 44.9 56.4 6.4 7.1 7.1 Z2.6 :E~ 2 37.9 Ll,3 5.7 3.1 1.7 6.7 16 1 63 85.711.3 4 22.6 125 :NA E. cloacae 1 16 1.4 2 4 4 5.? 44.9 ,-. . 2 2250 9.5 13.5 9.~ 37.9 26.9 32 :Ila~ . 2 2250 5.7 8 5.7 9.5 B 37.9 1 16 82. 8 4 1 ~ 16 . 2 ~250 166~7 4.8 2.8 19 37.9 :~A P. ~:tuartii2 32 22.69.5 8 . 9.5 3.4 32 2 32 13.5 13~5 ~.7 ~.~ 6.? 6.7 ~C+ 2 32 1611.36.7 1.4 8 11.3 5NA S. mArcescen~ 1 63 16 8 4 8 4 4 _ 1 2250 16 16 4 8 4 2.8 ~Na+ 1 63 4 8 4 2 2 11.3 1 2250 1L5 8 4 2 2.8 11.3 1 2250 63 11.3 2 :1 ~.8 ~.8 :NA S. aureun 3 3.6 4 4 6.4 11.3 44.9 125 :Na+ 2 4 2.8 1.2 5.7 16 5.7 88.7 51~+ 3 3.6 4 4.510.1 9 35.a 99.5 ` ~ 52~A S. faecsll~ 2 2.8 5.711.3 8 2177 125 ~250 a~ 1 2.8 32 4 8 125 16 2250 !
2 2.8 g.5 B 8 44.9 63 225û

et;ic r~n plue- (-~ co=bin-t~pp~ e p~ bl-~ ~ro~ 2 7~er~ e-5~

TABLE XVI

Jlntlbaceerial Act~rltJ of Chlorhex~dine Dlgluconate in Combln~tlon with Phosph~nillc ~d ~PA) or lts Na~ and ~ Salts .. ..... .
~ I C ~tlg/ml)~
Drug Comblnatlon~ Organls~ of Chlorhexidine:Phosphanilate (%:Z) StralQs 100:0 95:5 75:25 50:50 25:75 5:95 0:100 .... . ____ ChlorhexidlDe :PA P.aeruglnosa 10 17.8 5.2 3 l.g 1.5 2.3 1.9 Dl~luconate :Na~ 10 17.8 7.9 4.1 3.4 5.1 2.9 Z 3 :~ . 10 17,5 8.9 4.7 3.9 3 3,3 ~ 7 :PAE. coli . 0.4 0.5 0.5 0.5 1.2 6.4 2184 :Na+ 3 0.4 0.5 0.5 0.8 0.6 6.9 21~0 :1~ . . 3 . 0.4 1~.5 1~.4 0.9 0.6 7.4 ~157 :PAR. pDeumonlae 3 3.6 2.4 2.ô 4.5 5 67 2177 :Na+ 3 3.4 2.5 4 5.3 6.7 84 2l87 :ICf 3 3.6 3 3.~ 5.7 ~.5 48 2177 :PA1!. cloacae 3 2.7 2 2 1 3 6 4 2 45 2l32 :~a+ 3 2.7 2.2 3 4 4 8 6 4 50 U49 :1~ 3 2.7 2.4 3.2 4.5 9.5 56 2177 :PAP. aeuartil 2 1.5 1.4 lo7 1~7 1.5 3.7 5.7 ~Nal 2 1.5 1.5 2.2 2.6 3.1 5.7 9.5 2 1.5 2 ~.2 2.4 Z.6 6.2 9 :PAS. Ioarcesceus 2 8.7 3.1 2.2 1.3 1 1 0.8 la+ 2 8.7 3.7 2.8 2.8 2 1.4 O.g + : ~ 2 8.7 4.8 2.8 2.8 1.7 1.8 1.7 :PAS. aureus 3 0.3 0.4 0.3 0.4 0.7 5.4 2158 - ~: + . 3 0.3 0.5 0 4 0 5 1.7 J~.3 2358 : 3 0.3 0.4 0 3 0 5 0.9 5 184 :PA S. ~aecalls 2 0.6 0.6 0.6 0.8 2 9 . 198 ~Na~ . 2 0.6 0.6 O.g 1 3.2 9 2250 . ~ 2 0.6 0.6 0.6 0.7 1.8 11.. 3 225~ :
~ ~ .'....................... . ~ . I
4eDmetrlc ~an ~alUI!19 tof oDmlji~atior~, where ~ppllca'ole) from 3 t~ 5 e~periDIents.

:~: :, .
"

7~3~ Ei TABLE XVI I

., , I
A~tlbacterinl Activi~y of Chlorhex~dine Diace~nte in Co~binatlon with Phosphanilic Acld ~PA) sr l~s Na nnd R~ Salts No. ~ I C t~glnl~*
Drug Combinatlon~ Organis~ of ~hlorhexidlne:PhosphaDllate tZ:%) Straiss100:0 95:5 75:25 50:5D 25:75 5:95 0:100 _ _ ~ , Chlorhexidine :PA P.aerugiDosa 6 13.7 18 7.4 5.2 7.7 3.6 1.7 Diacetnte :Na~ 1 2.5 4 4 6.4 8 8 32 7 14.5 17.1 .7 5.9 3.6 3.2 1.3 : ~ 1 2.5 1.6 4 5 16 32 40.1 7 14.5 20.8 11.9 7.3 7 7.~ 1.7 P~ E coli 2 0.8 0.9 1.2 1.6 2.3 11.3 125 Na~ . 2 0.B 0.5 0.9 0.9 2.1 9.2 2154 , ~+ 2 0.8 0.5 0.8 1.1 3 11.3 2 89 :PA R. pneu~Dnlae 2 3.5 3.5 4.9 5.7 11.3 59 2 89 Na~ 2 4 4 7.3 8 19 82 ~193 ;~t 2 3.5 2.8 ~.6 10.~18.3 59 ~102 PA ~ cloacse 3 2.9 6.1 6.1 7-3 13.3 276 2165 Na~ . 3 3.6 3.8 6 4 18167 2210 ~R~ 3 2.9 3.3 4.4 ~.316.8 79 ~150 :PA P. ~tuartii 1 2.6 5.3 5.3 4.6 4.6 9.2 :Na~ 1 2.6 3 3.5 4.6 4 8 12.1 g~ i 2.6 2.3 3 3.510.6 14 10.6 :PA S ~arcescens 2 13 13 4.6 5.3 3 3.3 1.4 Na~ . 2 16 26.9 6.7 5.2 2.4 3.1 1.1 'R+ 2 13 21.1 7.5 5.7 6.1 J.5 5.7 :PA S. aureus 3 0.4 0.6 0.8 0.9 1.7 6.4 2 75 ;
:Na~ 3 0.4 0.4 0.5 0.6 1.2 4.B 214p : ~ 3 0.4 0.~ 0.6 0.8 1.7 7.1 ~ 79 PA S fnecalis 2 0.8 0.8 l.S 2.1 3 13.9 ~250 'N~l . 2 0.8 0.8 1.2 1.2 2.6 14.9 2250 R~ 2 0.8 0.7 1.2 1.6 4.371.3 2165 ~Geo~etrie ~ea~ values (of comblnattons, ~here nppllcable) fro~ 3 ~o 5 experIments.

. ~
, , - . . .

~7S3~Q~

TABLE XVI I I

Antlbacterlal Activ~ty of Chlorhexidine ~ydrochloride ln Co~bloatlon ~ith Phosphanillc Acld (PA~ Dr lt~ Na+ aDd K+ Salts No. ___ 21 I C tP~ )*
Dr~lg Go~biDatlon~ Or~aDis~ of Chlorhexidine:Phosphanilate (2:2) Strains 100:0 95:5 75:25 50:50 25:75 5 95 0 100 .._ . . ~ _ ChlorhexidlDe :PA p.aerugiDosa 2 24.224.2 21.1 26 39.2 51.4 ~59 Eiydrochloride ô 2125 14.9 5.12.8 2.4 1.9 2.3 :Na+ 1 36.6 27.9 27.9 27.9 42 48.1 55 7 ~105 21.5 84.6 3.6 2.2 2.8 :1~ 1 36.6 21.1- 4242 32 36.6 32 6 2125 27.9 9.66.2 2.8 2.4 2.5 .
:PA E.coLi 3 2.8 2.4 3 5 12.7 263 21Bl :Na+ 2 2.8 3.4 6.2 11.3 27 89 ~149 :K~ 3 2.8 2.8 7 7 10.6 32 2165 :PA R.pneu~oDiae 3 23.2 37.925.4 240 75 2140 2149 -:IJa+ 3 32 25.430.2 2892132 223 2223 -:1~ 3 23.2 45.9 242 24D287 181 2190 -,PA }~. cloacae 1 16 20.210.1 20.2 50.3 2250 :>250 :N~q+ 1 10.6 7 16 32263 144 2250 RT 1 10.6 9.2221.1 242272 144 2218 :PA P.8tuartii 2 14.7 14.7 7.3 9.5 ~2.3 13.5 16 Na+ 2 14.9 9.911.3 11.3 9.9 10.6 14.9 .~ 2 14.7 16 10.4 12.3 ô 10.4 20.8 :PA S. marcesceDs 1 24.310.55.3 3.5 3 3 2.6 1 2165 10.6 5.3 3.5 2.3 2.6 2.3 :Na~ 1 26.9 11.3 3 5.7 5.7 3.4 2.8 :R~ 1 24.3 18.~13.9 9.2 5.3 4 3 1 2165 24.313.9 8 4 3 5-3 :PA 5.Aurew 3 2.4 2.4 1.9 3.8 9 3S.8 2149 :1~ 2 2.3 1.6 3.3 4.9 8 45 2150 3 2.1 2.8 3.4 4.8 6.7 22.6 2198 :PA S.faecalia 2 4 2.8 4.4 7.3 1997 2250 ` :Na~ 2 4 3.5 6.1 9.9 17.2 89 2250 lC+ 2 2.8 3.4 8 la.4 13.5 49 ~210 ::

~ ~ *CeoDIetric laesn values tof co~biDatlons, ~here applicable~ frocl 3 to 5 experi~ents.

~ .
:

:~' ` ~L17S3S6 TABLE XIX

Mean Synergism Indices Against Bacteria of Chlorhexidine Salts in Combination with Nalidixic Acid or Its Salts Mean Synergism Index Chlorhexidine:Nalidixate (%:%) Chemical Combinations 95:5 75:25 50:50 25:75 5:95 Chlorhexidine digluconate wi-th Nalidixic Acid .54 .49 .42 .39 .60 Chlorhexidine digluconate with Sodium nalidixate .79 .58 .21 .30 .37 Chlorhexidine digluconate with Potassium nalidixate .~7 .71 .57 .66 .59 Chlorhexidine diacetate with Nalidixic acid 1.49 1.51 .40 .59 .74 Chlorhexidine diacetate with Sodium nalidixate 1.98 1.31 .99 .99 1.12 Chlorhexidine diacetate with Potassium nalidixate 2.39 1.45 1.23 1.20 1.60 CE~lorhexidine hydrochloride : with Nalidixic acid .~7 .92 .58 2.22 .79 : ~Chlorhexidine hydrochloride with Sodium nalidixate 1.46 .34 .57 1.70 .49 :: Chlorhexidine hydrochloride with Potassium nalidixate. .47 .71 .57 .73 .65 ::

.
., ~17~35~Si TABLE XX

Mean Synergism Indices Against Bacteria of Chlorhexidine Salts in Combination with Phosphanilic Acid or Its Salts Mean Synergism Index Chlorhexidine:Phosphanilate (h %) Chemical Combination 95:575:2550:5025:75 5:95 Chlorhexidine digluconate with Phosphanilic Acid .83.74 .68 .62 1.01 Chlorhexidine digluconate with Sodium phosphanilate .92.97 .99 1.07 1.10 Chlorhexidine digluconate with Potassium phosphanilate .95.79 .85 .69 .97 Chlorhexidine diacetate with Phosphanilic acid 1.4~1.38 1O33 1.38 1.44 Chlorhexidine.diacetate w;th _ Sod;um phosphanilate 1.301.26 1.23 1.14 1.44 Chlorhexidine diacetate with Potassium phosphanilate 1.051.16 1,17 1.58 1.59 Chlorhexidine hydrochloride .
with Phosphanilic acid .85.66 .78 .97 1.17 Chlorhexidine hydrochloride .
with Sodium phosphanilate .731.00 1.20 1.31 1.21 Chlorhexidine Hydrochloride with Potassium phosphanil a~ ¦ .99 1 .34 1.30 1 05 . 95 ., ~7S35~

Compositions having the ratio of 35 to 65%
of a pharmaceutically acceptable chlorhexidine salt and 65 - 35% of phosphanilic acid, a salt of phosphanilic acid, nalidixic acid or a salt of nalidixic acid are disclaimed from the claims, in view of Canadian Patent 1,148,863 issued June 28,1983.

~; ' ;

Claims (12)

1. A composition for inhibiting growth of a bacterial species comprising a mixture of (a) a chlor-hexidine salt and (b) phosphanilic acid, a salt of phos-phanilic acid, nalidixic acid, or a salt of nalidixic acid; components (a) and (b) being present in the mixture in respective weight percentage ratios such that the mixture has a synergistic antibacterial effect on said species but with the exclusion of the ratio 35:65% of (a) to 65:35%
of (b).

2. The composition, as claimed in claim 1, wherein the salt of phosphanilic acid and the salt of nalidixic acid is an alkali metal salt.

3. The composition, as claimed in claim 1, wherein the chlorhexidine salt is selected from the group consisting of chlorhexidine hydrochloride, chlorhexidine digluconate, and chlorhexidine diacetate.

4. The composition, as claimed in claim 3, wherein the salt of phosphanilic acid and the salt of nalidixic acid is an alkali metal salt.

5. The composition, as claimed in claim 4, wherein the alkali metal salt is the sodium or potassium salt.

6. The composition, as claimed in claim 1, wherein component (a) is chlorhexidine digluconate, component (b) is nalidixic acid, or a sodium or potassium salt of nalidixic acid, and components (a) and (b) are present in the mixture in a respective weight percentage ratio of about 95:5 to about 5:95.

7. The composition, as claimed in claim 1 , wherein component (a) is chlorhexidine digluconate, component (b) is potassium phosphanilate, and components (a) and (b) are present in the mixture in a respective weight percentage ratio of about 75:25 to about 25:75.

8. The composition, as claimed in claim 1, wherein component (a) is chlorhexidine digluconate, component (b) is phosphanilic acid, and components (a) and (b) are present in the mixture in a respective weight percentage ratio of about 95:5 to about 25:75.

9. The composition, as claimed in claim 1 , wherein component (a) is chlorhexidine hydrochloride, component (b) is phosphanilic acid and components (a) and (b) are present in the mixture in a respective weight percentage ratio of about 95:5 to about 50:50.

10. The composition, as claimed in claim 1, wherein component (a) is chlorhexidine diacetate, component (b) is nalidixic acid, and components (a) and (b) are present in the mixture in a respective weight percentage ratio of about 50:50 to about 5:95.

11. The composition, as claimed in claim 1, wherein component (a) is chlorhexidine hydrochloride, component (b) is potassium nalidixic acid, and components (a) and (b) are present in the mixture in a respective weight percentage ratio of about 75:25 to about 5:95.

12. A method for inhibiting the growth on an inanimate substrate of a microorganism sensitive thereto comprising the step of contacting the microorganism with an amount sufficient to inhibit its growth of a composition as in claim 1.